Biomedical Engineering Reference
In-Depth Information
a compact tissue (vertebrate brain) or needs to travel a long dis-
tance in a dendritic tree.
A number of studies have been conducted to determine the
molecular mechanisms that result in potential-dependent optical
properties. The available evidence supports three different mech-
anisms (for different dyes): Dipole rotation
(5)
, electrochromism
(2)
, and a potential sensitive monomer-dimer equilibrium
(4)
.In
many cases, it has been possible to show that pharmacological and
photodynamic effects are small (e.g.
(6-12)
).
1.2.
Calcium-Sensitive
Dyes
Figure 3.2B
shows the chemical structure of a calcium-sensitive
dye, Calcium Green-1, together with a plot of the fluorescence
spectrum as a function of the free calcium concentration. This
dye signal reaches 50% of its maximum at a calcium concentration
of about 0.2
M. In contrast to the voltage-sensitive dyes, the cal-
cium dyes are located intracellularly. The dye is presumed to be
in the cytoplasm and to report changes in the calcium concentra-
tion in the cytoplasm, although some of the dye may be in other
intracellular compartments. These dyes are slower to respond and,
because they also act as buffers of calcium, the calcium signal in
the presence of dye may substantially outlast the change in cal-
cium concentration that would occur in the absence of the dye
(13, 14)
.
We begin with examples obtained from measurements
addressing three quite different neurobiological problems. In
one example where the light level was low, the camera was
a fast, back-illuminated, CCD camera with 80
μ
80 pixel spa-
tial resolution. In the second example, cellular resolution could
only be obtained using two-photon scanning microscopy. In the
third example, a slower CCD camera with 256
×
256 pixel spa-
tial resolution was used. The optical signals in the three exam-
ples are not large, they represent fractional changes in light
intensity (
×
10
−
1
and have modest
signal-to-noise ratios. Nonetheless, they can be measured with
an acceptable signal-to-noise ratio after attention to details of
the measurement which are described in the second part of the
chapter.
Figure 3.3
illustrates the three qualitatively different applica-
tions in neurobiology where imaging has been useful. First (left
panel), in order to know how a neuron integrates its synaptic
input into its action potential output, one needs to be able to
measure membrane potential everywhere where synaptic input
occurs and at the places where spikes are initiated. Second (mid-
dle panel), in order to understand how a nervous system gen-
erates a behavior, it is important to measure the action poten-
tial activity of many of the participating neurons. Third (right
panel), responses to sensory stimuli and generation of motor out-
put in the vertebrate brain are often accompanied by synchronous
10
−
3
to 5
I/I) of from 5
×
×
